The underappreciated diversity of bile acid modifications.

The repertoire of modifications to bile acids and related steroidal lipids by host and microbial metabolism remains incompletely characterized. To address this knowledge gap, we created a reusable resource of tandem mass spectrometry (MS/MS) spectra by filtering 1.2 billion publicly available MS/MS spectra for bile-acid-selective ion patterns. Thousands of modifications are distributed throughout animal and human bodies as well as microbial cultures. We employed this MS/MS library to identify polyamine bile amidates, prevalent in carnivores. They are present in humans, and their levels alter with a diet change from a Mediterranean to a typical American diet. This work highlights the existence of many more bile acid modifications than previously recognized and the value of leveraging public large-scale untargeted metabolomics data to discover metabolites. The availability of a modification-centric bile acid MS/MS library will inform future studies investigating bile acid roles in health and disease.

Reverse metabolomics for the discovery of chemical structures from humans.

Determining the structure and phenotypic context of molecules detected in untargeted metabolomics experiments remains challenging. Here we present reverse metabolomics as a discovery strategy, whereby tandem mass spectrometry spectra acquired from newly synthesized compounds are searched for in public metabolomics datasets to uncover phenotypic associations. To demonstrate the concept, we broadly synthesized and explored multiple classes of metabolites in humans, including N-acyl amides, fatty acid esters of hydroxy fatty acids, bile acid esters and conjugated bile acids. Using repository-scale analysis1,2, we discovered that some conjugated bile acids are associated with inflammatory bowel disease (IBD). Validation using four distinct human IBD cohorts showed that cholic acids conjugated to Glu, Ile/Leu, Phe, Thr, Trp or Tyr are increased in Crohn's disease. Several of these compounds and related structures affected pathways associated with IBD, such as interferon-γ production in CD4+ T cells3 and agonism of the pregnane X receptor4. Culture of bacteria belonging to the Bifidobacterium, Clostridium and Enterococcus genera produced these bile amidates. Because searching repositories with tandem mass spectrometry spectra has only recently become possible, this reverse metabolomics approach can now be used as a general strategy to discover other molecules from human and animal ecosystems.

MDM2 and MDMX promote ferroptosis by PPARα-mediated lipid remodeling.

MDM2 and MDMX, negative regulators of the tumor suppressor p53, can work separately and as a heteromeric complex to restrain p53's functions. MDM2 also has pro-oncogenic roles in cells, tissues, and animals that are independent of p53. There is less information available about p53-independent roles of MDMX or the MDM2-MDMX complex. We found that MDM2 and MDMX facilitate ferroptosis in cells with or without p53. Using small molecules, RNA interference reagents, and mutant forms of MDMX, we found that MDM2 and MDMX, likely working in part as a complex, normally facilitate ferroptotic death. We observed that MDM2 and MDMX alter the lipid profile of cells to favor ferroptosis. Inhibition of MDM2 or MDMX leads to increased levels of FSP1 protein and a consequent increase in the levels of coenzyme Q10, an endogenous lipophilic antioxidant. This suggests that MDM2 and MDMX normally prevent cells from mounting an adequate defense against lipid peroxidation and thereby promote ferroptosis. Moreover, we found that PPARα activity is essential for MDM2 and MDMX to promote ferroptosis, suggesting that the MDM2-MDMX complex regulates lipids through altering PPARα activity. These findings reveal the complexity of cellular responses to MDM2 and MDMX and suggest that MDM2-MDMX inhibition might be useful for preventing degenerative diseases involving ferroptosis. Furthermore, they suggest that MDM2/MDMX amplification may predict sensitivity of some cancers to ferroptosis inducers.

Leptochelins A-C, Cytotoxic Metallophores Produced by Geographically Dispersed Leptothoe Strains of Marine Cyanobacteria.

Metals are important cofactors in the metabolic processes of cyanobacteria, including photosynthesis, cellular respiration, DNA replication, and the biosynthesis of primary and secondary metabolites. In adaptation to the marine environment, cyanobacteria use metallophores to acquire trace metals when necessary as well as to reduce potential toxicity from excessive metal concentrations. Leptochelins A-C were identified as structurally novel metallophores from three geographically dispersed cyanobacteria of the genus Leptothoe. Determination of the complex structures of these metabolites presented numerous challenges, but they were ultimately solved using integrated data from NMR, mass spectrometry and deductions from the biosynthetic gene cluster. The leptochelins are comprised of halogenated linear NRPS-PKS hybrid products with multiple heterocycles that have potential for hexadentate and tetradentate coordination with metal ions. The genomes of the three leptochelin producers were sequenced, and retrobiosynthetic analysis revealed one candidate biosynthetic gene cluster (BGC) consistent with the structure of leptochelin. The putative BGC is highly homologous in all three Leptothoe strains, and all possess genetic signatures associated with metallophores. Postcolumn infusion of metals using an LC-MS metabolomics workflow performed with leptochelins A and B revealed promiscuous binding of iron, copper, cobalt, and zinc, with greatest preference for copper. Iron depletion and copper toxicity experiments support the hypothesis that leptochelin metallophores may play key ecological roles in iron acquisition and in copper detoxification. In addition, the leptochelins possess significant cytotoxicity against several cancer cell lines.

Connecting metabolome and phenotype: recent advances in functional metabolomics tools for the identification of bioactive natural products.

Covering: 1995 to 2023Advances in bioanalytical methods, particularly mass spectrometry, have provided valuable molecular insights into the mechanisms of life. Non-targeted metabolomics aims to detect and (relatively) quantify all observable small molecules present in a biological system. By comparing small molecule abundances between different conditions or timepoints in a biological system, researchers can generate new hypotheses and begin to understand causes of observed phenotypes. Functional metabolomics aims to investigate the functional roles of metabolites at the scale of the metabolome. However, most functional metabolomics studies rely on indirect measurements and correlation analyses, which leads to ambiguity in the precise definition of functional metabolomics. In contrast, the field of natural products has a history of identifying the structures and bioactivities of primary and specialized metabolites. Here, we propose to expand and reframe functional metabolomics by integrating concepts from the fields of natural products and chemical biology. We highlight emerging functional metabolomics approaches that shift the focus from correlation to physical interactions, and we discuss how this allows researchers to uncover causal relationships between molecules and phenotypes.

Functional genomics and metabolomics advance the ethnobotany of the Samoan traditional medicine "matalafi".

The leaf homogenate of Psychotria insularum is widely used in Samoan traditional medicine to treat inflammation associated with fever, body aches, swellings, wounds, elephantiasis, incontinence, skin infections, vomiting, respiratory infections, and abdominal distress. However, the bioactive components and underlying mechanisms of action are unknown. We used chemical genomic analyses in the model organism Saccharomyces cerevisiae (baker's yeast) to identify and characterize an iron homeostasis mechanism of action in the traditional medicine as an unfractionated entity to emulate its traditional use. Bioactivity-guided fractionation of the homogenate identified two flavonol glycosides, rutin and nicotiflorin, each binding iron in an ion-dependent molecular networking metabolomics analysis. Translating results to mammalian immune cells and traditional application, the iron chelator activity of the P. insularum homogenate or rutin decreased proinflammatory and enhanced anti-inflammatory cytokine responses in immune cells. Together, the synergistic power of combining traditional knowledge with chemical genomics, metabolomics, and bioassay-guided fractionation provided molecular insight into a relatively understudied Samoan traditional medicine and developed methodology to advance ethnobotany.

Evaluation of Data-Dependent MS/MS Acquisition Parameters for Non-Targeted Metabolomics and Molecular Networking of Environmental Samples: Focus on the Q Exactive Platform.

Non-targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a widely used tool for metabolomics analysis, enabling the detection and annotation of small molecules in complex environmental samples. Data-dependent acquisition (DDA) of product ion spectra is thereby currently one of the most frequently applied data acquisition strategies. The optimization of DDA parameters is central to ensuring high spectral quality, coverage, and number of compound annotations. Here, we evaluated the influence of 10 central DDA settings of the Q Exactive mass spectrometer on natural organic matter samples from ocean, river, and soil environments. After data analysis with classical and feature-based molecular networking using MZmine and GNPS, we compared the total number of network nodes, multivariate clustering, and spectrum quality-related metrics such as annotation and singleton rates, MS/MS placement, and coverage. Our results show that automatic gain control, microscans, mass resolving power, and dynamic exclusion are the most critical parameters, whereas collision energy, TopN, and isolation width had moderate and apex trigger, monoisotopic selection, and isotopic exclusion minor effects. The insights into the data acquisition ergonomics of the Q Exactive platform presented here can guide new users and provide them with initial method parameters, some of which may also be transferable to other sample types and MS platforms.

ReDU: a framework to find and reanalyze public mass spectrometry data.

We present ReDU ( https://redu.ucsd.edu/ ), a system for metadata capture of public mass spectrometry-based metabolomics data, with validated controlled vocabularies. Systematic capture of knowledge enables the reanalysis of public data and/or co-analysis of one's own data. ReDU enables multiple types of analyses, including finding chemicals and associated metadata, comparing the shared and different chemicals between groups of samples, and metadata-filtered, repository-scale molecular networking.

Bacterial and Chemical Evidence of Coastal Water Pollution from the Tijuana River in Sea Spray Aerosol.

Roughly half of the human population lives near the coast, and coastal water pollution (CWP) is widespread. Coastal waters along Tijuana, Mexico, and Imperial Beach (IB), USA, are frequently polluted by millions of gallons of untreated sewage and stormwater runoff. Entering coastal waters causes over 100 million global annual illnesses, but CWP has the potential to reach many more people on land via transfer in sea spray aerosol (SSA). Using 16S rRNA gene amplicon sequencing, we found sewage-associated bacteria in the polluted Tijuana River flowing into coastal waters and returning to land in marine aerosol. Tentative chemical identification from non-targeted tandem mass spectrometry identified anthropogenic compounds as chemical indicators of aerosolized CWP, but they were ubiquitous and present at highest concentrations in continental aerosol. Bacteria were better tracers of airborne CWP, and 40 tracer bacteria comprised up to 76% of the bacteria community in IB air. These findings confirm that CWP transfers in SSA and exposes many people along the coast. Climate change may exacerbate CWP with more extreme storms, and our findings call for minimizing CWP and investigating the health effects of airborne exposure.

Iron Chaperone Poly rC Binding Protein 1 Protects Mouse Liver From Lipid Peroxidation and Steatosis.

BACKGROUND AND AIMS: Iron is essential yet also highly chemically reactive and potentially toxic. The mechanisms that allow cells to use iron safely are not clear; defects in iron management are a causative factor in the cell-death pathway known as ferroptosis. Poly rC binding protein 1 (PCBP1) is a multifunctional protein that serves as a cytosolic iron chaperone, binding and transferring iron to recipient proteins in mammalian cells. Although PCBP1 distributes iron in cells, its role in managing iron in mammalian tissues remains open for study. The liver is highly specialized for iron uptake, utilization, storage, and secretion. APPROACH AND RESULTS: Mice lacking PCBP1 in hepatocytes exhibited defects in liver iron homeostasis with low levels of liver iron, reduced activity of iron enzymes, and misregulation of the cell-autonomous iron regulatory system. These mice spontaneously developed liver disease with hepatic steatosis, inflammation, and degeneration. Transcriptome analysis indicated activation of lipid biosynthetic and oxidative-stress response pathways, including the antiferroptotic mediator, glutathione peroxidase type 4. Although PCBP1-deleted livers were iron deficient, dietary iron supplementation did not prevent steatosis; instead, dietary iron restriction and antioxidant therapy with vitamin E prevented liver disease. PCBP1-deleted hepatocytes exhibited increased labile iron and production of reactive oxygen species (ROS), were hypersensitive to iron and pro-oxidants, and accumulated oxidatively damaged lipids because of the reactivity of unchaperoned iron. CONCLUSIONS: Unchaperoned iron in PCBP1-deleted mouse hepatocytes leads to production of ROS, resulting in lipid peroxidation (LPO) and steatosis in the absence of iron overload. The iron chaperone activity of PCBP1 is therefore critical for limiting the toxicity of cytosolic iron and may be a key factor in preventing the LPO that triggers the ferroptotic cell-death pathway.

Magnetotactic Bacteria Accumulate a Large Pool of Iron Distinct from Their Magnetite Crystals.

Magnetotactic bacteria (MTB) are ubiquitous aquatic microorganisms that form intracellular nanoparticles of magnetite (Fe3O4) or greigite (Fe3S4) in a genetically controlled manner. Magnetite and greigite synthesis requires MTB to transport a large amount of iron from the environment. Most intracellular iron was proposed to be contained within the crystals. However, recent mass spectrometry studies suggest that MTB may contain a large amount of iron that is not precipitated in crystals. Here, we attempted to resolve these discrepancies by performing chemical and magnetic assays to quantify the different iron pools in the magnetite-forming strain Magnetospirillum magneticum AMB-1, as well as in mutant strains showing defects in crystal precipitation, cultivated at various iron concentrations. All results show that magnetite represents at most 30% of the total intracellular iron under our experimental conditions and even less in the mutant strains. We further examined the iron speciation and subcellular localization in AMB-1 using the fluorescent indicator FIP-1, which was designed for the detection of labile Fe(II). Staining with this probe suggests that unmineralized reduced iron is found in the cytoplasm and associated with magnetosomes. Our results demonstrate that, under our experimental conditions, AMB-1 is able to accumulate a large pool of iron distinct from magnetite. Finally, we discuss the biochemical and geochemical implications of these results.IMPORTANCE Magnetotactic bacteria (MTB) produce iron-based intracellular magnetic crystals. They represent a model system for studying iron homeostasis and biomineralization in microorganisms. MTB sequester a large amount of iron in their crystals and have thus been proposed to significantly impact the iron biogeochemical cycle. Several studies proposed that MTB could also accumulate iron in a reservoir distinct from their crystals. Here, we present a chemical and magnetic methodology for quantifying the iron pools in the magnetotactic strain AMB-1. Results showed that most iron is not contained in crystals. We then adapted protocols for the fluorescent Fe(II) detection in bacteria and showed that iron could be detected outside crystals using fluorescence assays. This work suggests a more complex picture for iron homeostasis in MTB than previously thought. Because iron speciation controls its fate in the environment, our results also provide important insights into the geochemical impact of MTB.

In vivo bioluminescence imaging of labile iron accumulation in a murine model of Acinetobacter baumannii infection.

Iron is an essential metal for all organisms, yet disruption of its homeostasis, particularly in labile forms that can contribute to oxidative stress, is connected to diseases ranging from infection to cancer to neurodegeneration. Iron deficiency is also among the most common nutritional deficiencies worldwide. To advance studies of iron in healthy and disease states, we now report the synthesis and characterization of iron-caged luciferin-1 (ICL-1), a bioluminescent probe that enables longitudinal monitoring of labile iron pools (LIPs) in living animals. ICL-1 utilizes a bioinspired endoperoxide trigger to release d-aminoluciferin for selective reactivity-based detection of Fe2+ with metal and oxidation state specificity. The probe can detect physiological changes in labile Fe2+ levels in live cells and mice experiencing iron deficiency or overload. Application of ICL-1 in a model of systemic bacterial infection reveals increased iron accumulation in infected tissues that accompany transcriptional changes consistent with elevations in both iron acquisition and retention. The ability to assess iron status in living animals provides a powerful technology for studying the contributions of iron metabolism to physiology and pathology.

Manganese causes neurotoxic iron accumulation via translational repression of amyloid precursor protein and H-Ferritin.

For more than 150 years, it is known that occupational overexposure of manganese (Mn) causes movement disorders resembling Parkinson's disease (PD) and PD-like syndromes. However, the mechanisms of Mn toxicity are still poorly understood. Here, we demonstrate that Mn dose- and time-dependently blocks the protein translation of amyloid precursor protein (APP) and heavy-chain Ferritin (H-Ferritin), both iron homeostatic proteins with neuroprotective features. APP and H-Ferritin are post-transcriptionally regulated by iron responsive proteins, which bind to homologous iron responsive elements (IREs) located in the 5'-untranslated regions (5'-UTRs) within their mRNA transcripts. Using reporter assays, we demonstrate that Mn exposure repressed the 5'-UTR-activity of APP and H-Ferritin, presumably via increased iron responsive proteins-iron responsive elements binding, ultimately blocking their protein translation. Using two specific Fe2+ -specific probes (RhoNox-1 and IP-1) and ion chromatography inductively coupled plasma mass spectrometry (IC-ICP-MS), we show that loss of the protective axis of APP and H-Ferritin resulted in unchecked accumulation of redox-active ferrous iron (Fe2+ ) fueling neurotoxic oxidative stress. Enforced APP expression partially attenuated Mn-induced generation of cellular and lipid reactive oxygen species and neurotoxicity. Lastly, we could validate the Mn-mediated suppression of APP and H-Ferritin in two rodent in vivo models (C57BL6/N mice and RjHan:SD rats) mimicking acute and chronic Mn exposure. Together, these results suggest that Mn-induced neurotoxicity is partly attributable to the translational inhibition of APP and H-Ferritin resulting in impaired iron metabolism and exacerbated neurotoxic oxidative stress. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Copper regulates cyclic-AMP-dependent lipolysis.

Cell signaling relies extensively on dynamic pools of redox-inactive metal ions such as sodium, potassium, calcium and zinc, but their redox-active transition metal counterparts such as copper and iron have been studied primarily as static enzyme cofactors. Here we report that copper is an endogenous regulator of lipolysis, the breakdown of fat, which is an essential process in maintaining body weight and energy stores. Using a mouse model of genetic copper misregulation, in combination with pharmacological alterations in copper status and imaging studies in a 3T3-L1 white adipocyte model, we found that copper regulates lipolysis at the level of the second messenger, cyclic AMP (cAMP), by altering the activity of the cAMP-degrading phosphodiesterase PDE3B. Biochemical studies of the copper-PDE3B interaction establish copper-dependent inhibition of enzyme activity and identify a key conserved cysteine residue in a PDE3-specific loop that is essential for the observed copper-dependent lipolytic phenotype.

Copper is an endogenous modulator of neural circuit spontaneous activity.

For reasons that remain insufficiently understood, the brain requires among the highest levels of metals in the body for normal function. The traditional paradigm for this organ and others is that fluxes of alkali and alkaline earth metals are required for signaling, but transition metals are maintained in static, tightly bound reservoirs for metabolism and protection against oxidative stress. Here we show that copper is an endogenous modulator of spontaneous activity, a property of functional neural circuitry. Using Copper Fluor-3 (CF3), a new fluorescent Cu(+) sensor for one- and two-photon imaging, we show that neurons and neural tissue maintain basal stores of loosely bound copper that can be attenuated by chelation, which define a labile copper pool. Targeted disruption of these labile copper stores by acute chelation or genetic knockdown of the CTR1 (copper transporter 1) copper channel alters the spatiotemporal properties of spontaneous activity in developing hippocampal and retinal circuits. The data identify an essential role for copper neuronal function and suggest broader contributions of this transition metal to cell signaling.

An Endoperoxide Reactivity-Based FRET Probe for Ratiometric Fluorescence Imaging of Labile Iron Pools in Living Cells.

Iron is essential for sustaining life, as its ability to cycle between multiple oxidation states is critical for catalyzing chemical transformations in biological systems. However, without proper regulation, this same redox capacity can trigger oxidative stress events that contribute to aging along with diseases ranging from cancer to cardiovascular and neurodegenerative disorders. Despite its importance, methods for monitoring biological iron bound weakly to cellular ligands-the labile iron pool-to generate a response that preserves spatial and temporal information remain limited, owing to the potent fluorescence quenching ability of iron. We report the design, synthesis, and biological evaluation of FRET Iron Probe 1 (FIP-1), a reactivity-based probe that enables ratiometric fluorescence imaging of labile iron pools in living systems. Inspired by antimalarial natural products and related therapeutics, FIP-1 links two fluorophores (fluorescein and Cy3) through an Fe(II)-cleavable endoperoxide bridge, where Fe(II)-triggered peroxide cleavage leads to a decrease in fluorescence resonance energy transfer (FRET) from the fluorescein donor to Cy3 acceptor by splitting these two dyes into separate fragments. FIP-1 responds to Fe(II) in aqueous buffer with selectivity over competing metal ions and is capable of detecting changes in labile iron pools within living cells with iron supplementation and/or depletion. Moreover, application of FIP-1 to a model of ferroptosis reveals a change in labile iron pools during this form of cell death, providing a starting point to study iron signaling in living systems.

Recognition- and reactivity-based fluorescent probes for studying transition metal signaling in living systems.

Metals are essential for life, playing critical roles in all aspects of the central dogma of biology (e.g., the transcription and translation of nucleic acids and synthesis of proteins). Redox-inactive alkali, alkaline earth, and transition metals such as sodium, potassium, calcium, and zinc are widely recognized as dynamic signals, whereas redox-active transition metals such as copper and iron are traditionally thought of as sequestered by protein ligands, including as static enzyme cofactors, in part because of their potential to trigger oxidative stress and damage via Fenton chemistry. Metals in biology can be broadly categorized into two pools: static and labile. In the former, proteins and other macromolecules tightly bind metals; in the latter, metals are bound relatively weakly to cellular ligands, including proteins and low molecular weight ligands. Fluorescent probes can be useful tools for studying the roles of transition metals in their labile forms. Probes for imaging transition metal dynamics in living systems must meet several stringent criteria. In addition to exhibiting desirable photophysical properties and biocompatibility, they must be selective and show a fluorescence turn-on response to the metal of interest. To meet this challenge, we have pursued two general strategies for metal detection, termed "recognition" and "reactivity". Our design of transition metal probes makes use of a recognition-based approach for copper and nickel and a reactivity-based approach for cobalt and iron. This Account summarizes progress in our laboratory on both the development and application of fluorescent probes to identify and study the signaling roles of transition metals in biology. In conjunction with complementary methods for direct metal detection and genetic and/or pharmacological manipulations, fluorescent probes for transition metals have helped reveal a number of principles underlying transition metal dynamics. In this Account, we give three recent examples from our laboratory and collaborations in which applications of chemical probes reveal that labile copper contributes to various physiologies. The first example shows that copper is an endogenous regulator of neuronal activity, the second illustrates cellular prioritization of mitochondrial copper homeostasis, and the third identifies the "cuprosome" as a new copper storage compartment in Chlamydomonas reinhardtii green algae. Indeed, recognition- and reactivity-based fluorescent probes have helped to uncover new biological roles for labile transition metals, and the further development of fluorescent probes, including ones with varied Kd values and new reaction triggers and recognition receptors, will continue to reveal exciting and new biological roles for labile transition metals.

Synthetic fluorescent probes for studying copper in biological systems.

The potent redox activity of copper is required for sustaining life. Mismanagement of its cellular pools, however, can result in oxidative stress and damage connected to aging, neurodegenerative diseases, and metabolic disorders. Therefore, copper homeostasis is tightly regulated by cells and tissues. Whereas copper and other transition metal ions are commonly thought of as static cofactors buried within protein active sites, emerging data points to the presence of additional loosely bound, labile pools that can participate in dynamic signalling pathways. Against this backdrop, we review advances in sensing labile copper pools and understanding their functions using synthetic fluorescent indicators. Following brief introductions to cellular copper homeostasis and considerations in sensor design, we survey available fluorescent copper probes and evaluate their properties in the context of their utility as effective biological screening tools. We emphasize the need for combined chemical and biological evaluation of these reagents, as well as the value of complementing probe data with other techniques for characterizing the different pools of metal ions in biological systems. This holistic approach will maximize the exciting opportunities for these and related chemical technologies in the study and discovery of novel biology of metals.